Recent advances in analytical separation techniques for therapeutic oligonucleotides.

Therapeutic oligonucleotides are an emerging class of drugs designed for gene expression modulation. The increasing number of clinical trials and currently expanding market is facilitating further integration and accessibility of these therapeutics. A crucial step in drug development involves reliable analytical tools for characterization and quality control. For clinical applications, oligonucleotides must be separated and purified to ensure regulatory compliance. However, their analysis represents a complex bioanalytical challenge, grounded in their complex impurity profiles. Chemical stability and binding affinity of oligonucleotide-based therapeutics are enhanced during synthesis by extensive modifications, inducing formation of various synthesis failures or truncated sequences. Furthermore, meeting current guidelines or addressing manufacturing scale-up strategies remains challenging as each oligonucleotide typically necessitates a custom analytical protocol. Here, we provide an overview of the most recent advances in separation methods, including various chromatography methods and capillary electrophoresis for nucleic acid-based therapeutics.